Figuring out how cellular molecules work in real time and space has been
hampered by a kind of uncertainty principle. Tissues can be imaged while
intact—but with preparations that prevent molecular phenotyping. Or they can
be labeled—but then imaged only in sections.

Thus Karl Deisseroth, who holds a dual appointment in Stanford’s Department of
Bioengineering and Department of Psychiatry and Behavioral Sciences, and his
team “set ourselves the goal of rapidly transforming intact tissue into an
optically transparent and macromolecule permeable construct while
simultaneously preserving native molecular information and structure.”
Their results were published in Nature (1).

Mouse brains have been made transparent before, most notably by using the SCALE
method developed in 2011 by Japanese researchers. But CLARITY is
different, said Deisseroth, because it allows “not just transparency but
labeling within the transparent organ in a very flexible, versatile way. You
can use any molecular label to paint different neurons different colors and
see which are excitatory and which are inhibitory. CLARITY can add a lot of
information that was not available before.”

CLARITY replaces the structural function of the cell membrane’s lipid
bilayer—which effectively keeps useful reagents like antibodies, fluorescent
dyes, and photons out of cells—with a formaldehyde crosslinked hydrogel
mesh. The hydrogel is permeable to light and macromolecules, but physically
supports the tissue structure by chemically incorporating native
biomolecules. Lipids and other molecules that remain unbound are removed to
reduce light scattering.

Using this method, it took the researchers about a week to clear the tissue in
an adult mouse brain. They then imaged the entire 5-6-mm thick translucent
mouse brain at cellular resolution using single-photon microscopy.
Structural features like synapses and dendritic spines remained in place;
labeled fibers were easily seen in the neocortex, nucleus accumbens,
caudate-putamen, and amygdala.

In contrast to other fixing methods, CLARITY retains about 92% of native
proteins, so the brain can be subjected to repeated labeling; the group
demonstrated that at least three rounds of labeling can be performed
successfully. And since CLARITY’s hydrogel both increases tissue
permeability and decreases light scattering relative to cell membranes,
molecular probes can diffuse deep into, and be detected deep inside, intact
tissue. Diesseroth and colleagues believe that the technique might one day
be compatible with electron microscopy, allowing more detailed imaging.

In addition to the mouse brain, the group clarified 0.5-mm blocks of a human
brain that had been stored in formalin for over six years. The blocks were
from the frontal lobe of an autistic patient, and staining revealed
structural abnormalities in deep layers of the tissue that were absent in
normal age- and sex- matched controls.

“The brain is the most difficult organ, because it has the most dense areas of
lipid interfaces and that is what blocks light,” said Deisseroth. “We don’t
anticipate serious problems with other organs, although the methodology
might have to be modified somewhat.” Already, cancer researchers have
contacted him about clarifying tumor biopsies.

Deisseroth was one of the scientists who proposed and supports the recently
announced BRAIN
Initiative. “CLARITY arose because of interactions between
neuroscientists and chemical engineers—groups that do not normally speak to
each other,” he noted. “This new project will also get people talking.
Imagine the opportunities and new ideas it will bring to light.”